Sentinel Signal for Adaptive Retention Time in Targeted MS Methods

ABSTRACT

A plurality of MRM transitions to be used to monitor a sample are received and divided into two or more contiguous groups. At least one sentinel transition is selected in each group that identifies a next group of the two or more contiguous groups that is to be monitored. A first group of the two or more contiguous groups is placed on a duty cycle list of the tandem mass spectrometer. One or more compounds are separated from the sample and ionized, producing an ion beam. A series of MRM transitions read from the duty cycle list are executed on the ion beam by the tandem mass spectrometer. When at least one sentinel transition of the first group is detected, a next group identified by the sentinel transition is placed on the list.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 62/261,498, filed Dec. 1, 2015, the content ofwhich is incorporated by reference herein in its entirety.

INTRODUCTION

Various embodiments relate to the analysis of substances with a massspectrometer. In particular, various embodiments relate to the detectionof and relative or absolute quantification of substances by massspectrometry coupled to hyphenated techniques. Successive groups ofsubstances of interest are monitored as a function of successivedetection of signals, for example.

The coupling between a separation system and a mass spectrometer hasbeen used for many years for the detection and quantification ofsubstances in complex mixtures. Just as a spectrophotometer, forexample, the mass spectrometer can be used as a selective detector ofsubstances separated by chromatography or capillary electrophoresis.Substances detected can be small organic molecules such as drugs, dopingmolecules, pesticides, metabolites, proteins, or peptides, for example.In general, methods of detection and quantification using a couplingbetween a mass spectrometer and a separation technique use afragmentation step (tandem mass spectrometry, mass spectrometry—massspectrometry (MS-MS), MS2, MSn). Usually, the fragmentation is obtainedby a process of collision between ions and an inert gas but may alsoresult from interaction with electrons, photons, or a surface. Thisfragmentation step is sought to ensure the highest possible level ofdetection specificity thanks to the combination of information betweenthe intact species and the relative intensities of its fragments orproduct ions. The second level of specificity is provided by therelative or absolute retention time of the substance in the separationprocess.

The most common approach to perform detection and/or quantification of asubstance by mass spectrometry is the so-called targeted approach.Though any instrument allowing MSn experiment is suitable, the targetedmethod is mainly performed on a triple quadrupole mass spectrometerwhich results from the assembly of three quadrupoles. In this mode ofuse, the mass spectrometer is set to record the signal associated withan event called a transition where a precursor ion (often correspondingto the whole substance) is filtered in a first quadrupole thenfragmented in quadrupole 2 and one or successively many product ions arefiltered in the quadrupole 3. It is important to note here that thesignal associated with a transition is more intense when the massspectrometer remains on the observation of this transition.

When multiple transitions are observed for the same molecule, and evenmore importantly when multiple transitions are observed for severalmolecules to quantify, it is necessary to adjust the observation time toensure perfect definition of the shape of the chromatographic orelectrophoretic peaks. It is agreed that a minimum of 10 measurementpoints is typically required to draw such a peak to ensure thequantification accuracy and satisfactory accuracy.

Thus, if a chromatographic peak has a baseline width of 10 seconds andthe observation time (dwell time) of a transition is 10 milliseconds,then it will be possible to monitor up to 100 transitions since thetotal observation time thereof is 100×10 milliseconds=1 s. If twotransitions must be observed by molecules, no more than 50 molecules canbe detected and quantified during analysis, or 100/n if n moleculestransitions to be observed by molecules.

In very many situations, whether basic or applied research, it isdesirable to observe hundreds of substances to be quantified. This is,for example, the case in the detection of and quantification ofpesticides in environmental matrices (water and soil) or in food or inbiological fluids. Another well-known context is the targeted detectionand quantification of hundreds to thousands peptides in proteomics-basedapproaches. This context can be clinical evaluation studies of biomarkercandidates or the more fundamental context of systems biology. In thiscase, the limit imposed by the minimum number of measurement points toproperly define the shape of a peak needs to be circumvented by limitingthe observation time of a transition. Thus, instead of programming themonitoring of a transition for the duration of the separation method, itis followed only in a limited time window. This observation time windowcan be relatively large and gather the necessary transitions followed byseveral molecules. The optimal and often retained strategy is, however,to record the specific transitions of a substance only around itsretention time window. Different trade names refer to this process:scheduled multiple reaction monitoring (MRM) by AB Sciex, timed MRM byThermo, dynamic MRM by Agilent, for example. However, multiple causescan lead to a drift of retention time. The cause can be voluntary when,for example, in liquid chromatography the experimenter changes the slopeof the gradient, the column length, the composition of the mobile phase,or the particle diameter. The cause may also be independent of theexperimenter and results from column overloading, partial blockage ofthe column, temperature variation of the mobile phase.

Hence, any variation in retention time increases the possibility thatthe molecule is not detected in its programmed retention time window.Thus, to take into account these potential variations of retentiontimes, the width of the observation window is always wider than thewidth of the elution peak of the substance, the more often a factor oftwo to three. This precaution has the effect of restricting the numberof transitions and therefore the observable substances in a given unitof time. Similarly, the width of these windows should be reconsideredwhen the flow rate of the mobile phase will be radically changed bypassing from a conventional HPLC mode to ultra HPLC mode, or vice versa.Finally, the timing of the observation windows should also be checked assoon as the experimenter changes the stationary phase, because, even fora similar type of octadecyl (C18) graft, the grafting chemistry and thechemical nature of the beads affect the selectivity of the column thatcan result in retention time inversion.

Finally, another drawback of conventional methods is that they cannot beeasily transferred to another instrument or analysis site withoutperforming a first adjustment of the experiment retention time.

Recently, new methods have been introduced to facilitate the transfermethod of a device to another or between different sites. The firstmethod proposed by Escher et al. describes a method for normalizing thepeptide retention time against other reference peptides in order toassign them a universal retention time index. The claimed advantage ofthis method (iRT) is that it can better predict the empirical retentiontimes of peptides compared to a prediction algorithm. The process ishowever limited in that a first calibration step is always necessarywhen the method is transferred to another system. Also, this processdoes not significantly reduce the width of the detection windows, whichremains at a minute in most studies using this method. Recently, Domonet al. have proposed a method for improving the preceding iRT strategyfor on-the-fly correction of retention time. In this method, ideallyseveral compounds (peptides in this study) are used as anchors anddistributed uniformly throughout the chromatogram in order to detectpossible retention time drifts. In this method, two anchors elutedconsecutively are used to recalculate a possible correction of thegradient slope. This process can avoid changing the observation timewindows during a voluntary change in the gradient slope or flow, or whena variable dead volume causes a delay more or less in the elution of thefirst compound. However, this method has several limitations. It cannotbe applied in the case of a non-linear distortion of the gradientcaused, for example, by a transient rise in pressure. The correctionoccurs only when the second anchor is detected, so the elutedsubstances, before this second anchor, may not be detected if itsretention time moves. Finally, this method still requires the use ofprogrammed detection windows. Thus, in the study described by Domon etal., detection windows at least on the order of one or two minutes areused. In the study described by Guo et al., even larger detectionwindows were used for complex cellular extracts.

Thus, state of the art methods used to increase the multiplexingcapabilities of relative or absolute assay based on a separation systemcoupled to a mass spectrometer systematically are generally of twotypes. In the first type of method time segments are used where specifictransitions of eluted substances between two retention times areprogrammed. In the second type of method, a series of overlapping timewindows framing the retention times of the targeted substances are used.These windows are distributed according to the chronological elutionorder of the targeted substances.

It is therefore the object of this disclosure to provide an alternativemethod for the analysis of multiple substances in complex mixtures.

SUMMARY

At the start of the analysis, the mass spectrometer is set to detect afirst group i of transitions that contains a sentinel signal i. Thedetection of the sentinel signal i in the group of transitions i,considering the i signal is defined by one or more transitions above apredetermined threshold, opens a new group i+1 of transitions whichitself contains a sentinel signal i+1, which, when it is detected, inturn will open up a new group i+2 transitions which itself contains asentinel i+2 signal. In a group of transitions, several transitioncharacteristics of substances eluted immediately prior to a sentinelsignal are present in the next group to ensure continuity of detectingsubstances until they are completely eluted. Therefore, electrophoreticor chromatographic peaks are perfectly defined.

Within each group, the transitions can be arranged in a completelyrandom order as the number of transitions within a group is onlyconditioned by the difference of elution time width between two sentinelsignals.

Thus, the Sentinel process is radically different from all conventionalmethods, since this process does not use any time window to observetransitions, i.e., neither time window defined by two empirical orcalculated indexed retention time, nor a time window corrected in realtime.

These and other features of the applicant's teachings are set forthherein.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1 is a block diagram that illustrates a computer system, upon whichembodiments of the present teachings may be implemented.

FIG. 2 is an exemplary total ion current (TIC) chromatogram plot showinghow MRM transitions are grouped based on their corresponding expectedproduct ion chromatographic peaks, in accordance with variousembodiments.

FIG. 3 is an exemplary plot of a gradient of separation, in accordancewith various embodiments.

FIG. 4 is an exemplary TIC chromatogram plot showing the product ionpeaks produced by a method that used six sentinel transitions to triggerother groups of transitions for a sample that was separated according tothe gradient of separation shown in FIG. 3, in accordance with variousembodiments.

FIG. 5 is an exemplary plot of a gradient of separation that has anincreased slope in comparison to the gradient of separation shown inFIG. 3, in accordance with various embodiments.

FIG. 6 is an exemplary TIC chromatogram plot showing the product ionpeaks produced by a method that used the same six sentinel transitionsusing to produce FIG. 4 to trigger other groups of transitions for asample that was separated according to the gradient of separation shownin FIG. 5, in accordance with various embodiments.

FIG. 7 is a schematic diagram of system for triggering a group ofmultiple reaction monitoring (MRM) transitions from a series ofcontiguous groups when at least one sentinel transition of the group isdetected as part of a previous group, in accordance with variousembodiments.

FIG. 8 is a flowchart showing a method for triggering a group of MRMtransitions from a series of contiguous groups when at least onesentinel transition of the group is detected as part of a previousgroup, in accordance with various embodiments.

FIG. 9 is a schematic diagram of a system that includes one or moredistinct software modules that performs a method for triggering a groupof MRM transitions from a series of contiguous groups when at least onesentinel transition of the group is detected as part of a previousgroup, in accordance with various embodiments.

Before one or more embodiments of the present teachings are described indetail, one skilled in the art will appreciate that the presentteachings are not limited in their application to the details ofconstruction, the arrangements of components, and the arrangement ofsteps set forth in the following detailed description or illustrated inthe drawings. Also, it is to be understood that the phraseology andterminology used herein is for the purpose of description and should notbe regarded as limiting.

DESCRIPTION OF VARIOUS EMBODIMENTS Computer-Implemented System

FIG. 1 is a block diagram that illustrates a computer system 100, uponwhich embodiments of the present teachings may be implemented. Computersystem 100 includes a bus 102 or other communication mechanism forcommunicating information, and a processor 104 coupled with bus 102 forprocessing information. Computer system 100 also includes a memory 106,which can be a random access memory (RAM) or other dynamic storagedevice, coupled to bus 102 for storing instructions to be executed byprocessor 104. Memory 106 also may be used for storing temporaryvariables or other intermediate information during execution ofinstructions to be executed by processor 104. Computer system 100further includes a read only memory (ROM) 108 or other static storagedevice coupled to bus 102 for storing static information andinstructions for processor 104. A storage device 110, such as a magneticdisk or optical disk, is provided and coupled to bus 102 for storinginformation and instructions.

Computer system 100 may be coupled via bus 102 to a display 112, such asa cathode ray tube (CRT) or liquid crystal display (LCD), for displayinginformation to a computer user. An input device 114, includingalphanumeric and other keys, is coupled to bus 102 for communicatinginformation and command selections to processor 104. Another type ofuser input device is cursor control 116, such as a mouse, a trackball orcursor direction keys for communicating direction information andcommand selections to processor 104 and for controlling cursor movementon display 112. This input device typically has two degrees of freedomin two axes, a first axis (i.e., x) and a second axis (i.e., y), thatallows the device to specify positions in a plane.

A computer system 100 can perform the present teachings. Consistent withcertain implementations of the present teachings, results are providedby computer system 100 in response to processor 104 executing one ormore sequences of one or more instructions contained in memory 106. Suchinstructions may be read into memory 106 from another computer-readablemedium, such as storage device 110. Execution of the sequences ofinstructions contained in memory 106 causes processor 104 to perform theprocess described herein. Alternatively hard-wired circuitry may be usedin place of or in combination with software instructions to implementthe present teachings. Thus implementations of the present teachings arenot limited to any specific combination of hardware circuitry andsoftware.

In various embodiments, computer system 100 can be connected to one ormore other computer systems, like computer system 100, across a networkto form a networked system. The network can include a private network ora public network such as the Internet. In the networked system, one ormore computer systems can store and serve the data to other computersystems. The one or more computer systems that store and serve the datacan be referred to as servers or the cloud, in a cloud computingscenario. The one or more computer systems can include one or more webservers, for example. The other computer systems that send and receivedata to and from the servers or the cloud can be referred to as clientor cloud devices, for example.

The term “computer-readable medium” as used herein refers to any mediathat participates in providing instructions to processor 104 forexecution. Such a medium may take many forms, including but not limitedto, non-volatile media, volatile media, and transmission media.Non-volatile media includes, for example, optical or magnetic disks,such as storage device 110. Volatile media includes dynamic memory, suchas memory 106. Transmission media includes coaxial cables, copper wire,and fiber optics, including the wires that comprise bus 102.

Common forms of computer-readable media or computer program productsinclude, for example, a floppy disk, a flexible disk, hard disk,magnetic tape, or any other magnetic medium, a CD-ROM, digital videodisc (DVD), a Blu-ray Disc, any other optical medium, a thumb drive, amemory card, a RAM, PROM, and EPROM, a FLASH-EPROM, any other memorychip or cartridge, or any other tangible medium from which a computercan read.

Various forms of computer readable media may be involved in carrying oneor more sequences of one or more instructions to processor 104 forexecution. For example, the instructions may initially be carried on themagnetic disk of a remote computer. The remote computer can load theinstructions into its dynamic memory and send the instructions over atelephone line using a modem. A modem local to computer system 100 canreceive the data on the telephone line and use an infra-red transmitterto convert the data to an infra-red signal. An infra-red detectorcoupled to bus 102 can receive the data carried in the infra-red signaland place the data on bus 102. Bus 102 carries the data to memory 106,from which processor 104 retrieves and executes the instructions. Theinstructions received by memory 106 may optionally be stored on storagedevice 110 either before or after execution by processor 104.

In accordance with various embodiments, instructions configured to beexecuted by a processor to perform a method are stored on acomputer-readable medium. The computer-readable medium can be a devicethat stores digital information. For example, a computer-readable mediumincludes a compact disc read-only memory (CD-ROM) as is known in the artfor storing software. The computer-readable medium is accessed by aprocessor suitable for executing instructions configured to be executed.

The following descriptions of various implementations of the presentteachings have been presented for purposes of illustration anddescription. It is not exhaustive and does not limit the presentteachings to the precise form disclosed. Modifications and variationsare possible in light of the above teachings or may be acquired frompracticing of the present teachings. Additionally, the describedimplementation includes software but the present teachings may beimplemented as a combination of hardware and software or in hardwarealone. The present teachings may be implemented with bothobject-oriented and non-object-oriented programming systems.

Triggering Contiguous Groups of Transitions

Systems and methods for triggering a group of multiple reactionmonitoring (MRM) transitions from a series of contiguous groups when atleast one sentinel transition of the group is detected as part of aprevious group are described in this detailed description of theinvention,. In this detailed description, for purposes of explanation,numerous specific details are set forth to provide a thoroughunderstanding of embodiments of the present invention. One skilled inthe art will appreciate, however, that embodiments of the presentinvention may be practiced without these specific details. In otherinstances, structures and devices are shown in block diagram form.Furthermore, one skilled in the art can readily appreciate that thespecific sequences in which methods are presented and performed areillustrative and it is contemplated that the sequences can be varied andstill remain within the spirit and scope of embodiments of the presentinvention.

As described above, one targeted approach to the detection and/orquantification of a substance by mass spectrometry is called multiplereaction monitoring (MRM). MRM can also be referred to as selectedreaction monitoring (SRM). In these methods, precursor ion and production pairs, called transitions, which are used to identify or quantifyknown compounds, are predefined for a particular experiment. A tandemmass spectrometer is then used to repeatedly interrogate each of thesetransitions as compounds are separated from a mixture using a separationdevice. The tandem mass spectrometer interrogates each transition byselecting and fragmenting the precursor ion of the transition and thenanalyzing the resulting fragments for the intensity of the product ionof the transition. The tandem mass spectrometer reads the predefinedtransitions from list, for example. The list can be called a duty cyclelist, for example. The tandem mass spectrometer monitors each transitionof the predefined transitions during each cycle of a plurality of timecycles. The completion of the plurality of time cycles can be referredto as one acquisition.

Each transition meant to identify or quantify a particular knowncompound must be monitored while the known compound is separating oreluting. In addition, in order to provide enough resolution to identifyor quantify the known compound, the transition must be monitored manytimes while the known compound is separating or eluting. In other words,each separation of the known compound occurs over time and has anintensity versus time shape that is referred to a separation peak orelution peak. In order to provide enough resolution to identify orquantify each known compound, each transition must be monitored atenough times or data points across the elution peak. If each transitionis monitored at enough times or data points across the elution peak, aproduct ion peak with a similar shape to the elution peak is traced outfor the transition. This product ion peak is then used to identify orquantify the compound.

In addition, more than one transition may be need to identify orquantify a known compound. As a result, many transitions may bemonitored many times during each elution peak. However, since the tandemmass spectrometer takes time to monitor each transition, only a finitenumber of MRM transitions can be monitored during each elution peak.

The mass spectrometry industry has developed various methods to improveor maximize the number of MRM transitions that can be detected in oneanalytical workflow or during one acquisition. Currently, for example,thousands of different MRM transitions can be handled in a singleanalytical workflow or acquisition.

As described above, one exemplary method to improve or maximize thenumber of MRM transitions that one could detect in an analyticalworkflow is called scheduled MRM. In scheduled MRM, each MRM transitiondefined in the workflow has a retention time associated it.Consequently, each MRM transition is monitored only around its retentiontime. Therefore by scheduling the MRM transitions, the maximum number oftransitions that are monitored at any point in time during anacquisition is optimized. In other words, not all MRM transitions needto be monitored for the entire acquisition time. This approach providesmore data points across an elution peak and, therefore, betterprecision, sensitivity, and accuracy.

However, scheduled MRM has an important limitation. It is dependent onthe accuracy and absolute value of the retention time used for eachtransition. Whenever the separation device changes or the gradient ofseparation changes, the retention time for each transition must berecomputed. This becomes particularly cumbersome when workflows includethousands of MRM transitions. This also makes it difficult to usescheduled MRM workflows across separation devices produced by differentmanufacturers that have different elution rates.

In various embodiments, systems and methods are provided to limit thenumber of MRM transitions monitored at any one time without requiringthe re-computation of retention time for each MRM transition, wheneverthe separation device changes or the gradient of separation changes. Inthese systems and methods, the MRM transitions to be used for an entireacquisition are ordered according to an expected retention time. Theordered MRM transitions are then divided into contiguous groups withdifferent expected retention time ranges. In each group, at least onetransition is selected as a sentinel transition. The sentinel transitionin each group is used to identify the next group and trigger it formonitoring.

During acquisition, a first group of transitions is selected formonitoring. This is, for example, the group with the earliest expectedretention time. When at least one sentinel transition in the first groupis detected by the tandem mass spectrometer, the next group oftransitions identified by the at least one sentinel transition is addedto the list of transitions monitored by the tandem mass spectrometer. Inother words, at least one sentinel transition in each group is used totrigger the transitions in the next contiguous group.

A group of transitions can also be removed from monitoring. For example,once at least one sentinel transition in the next contiguous group isdetected, the transitions in the first group can be removed frommonitoring.

As a result, by using sentinel transitions to trigger the addition andsubtraction of MRM transitions from monitoring the overall number of MRMtransitions being monitored at any one time is reduced. In addition,because the groups of transitions are not dependent on a specificretention time, workflows based on these systems and methods can be usedwithout modification whenever the separation device changes or thegradient of separation changes.

FIG. 2 is an exemplary total ion current (TIC) chromatogram plot 200showing how MRM transitions are grouped based on their correspondingexpected product ion chromatographic peaks, in accordance with variousembodiments. In plot 200 the expected intensity versus retention timechromatographic peaks of 22 product ions are plotted. These 22 production peaks correspond to 22 MRM transitions (not show). The 22 production peaks are plotted in plot 200 as a TIC chromatogram and are,therefore, ordered according to increasing retention time. The production peaks, in turn, order the corresponding MRM transitions by retentiontime. The actual or absolute value of the retention times is notimportant. It is the order that is important.

Once the 22 MRM transitions are ordered based on the 22 product ionpeaks, the MRM transitions are grouped. In practice, groups are created,for example, by including in each group or across two groups the maximumnumber of MRM transitions that can be monitored with the desiredresolution across an elution peak. This maximum number could be 200 MRMtransitions, for example. In other words, the purpose of the groups isto limit the number of MRM transitions being monitored at any one timein order to maintain the resolution necessary across any one elutionpeak. After meeting this requirement, the groups can be selected in anyway. The groups, however, also have to be contiguous to ensure that allMRM transitions are monitored at some point.

For purposes of illustration here, the 22 MRM transitions correspondingto the 22 product ion peaks are grouped more or less evenly intoearly-eluting, mid-eluting, and late-eluting groups. For example, group211 includes the early-eluting product ion peaks, group 212 includes themid-eluting product ion peaks, and group 213 includes the late-elutingproduct ion peaks. Groups 211-213 are contiguous.

Groups 211-213, as shown, can also include overlapping regions. If aprevious group is removed as soon as a next group is added, theoverlapping product ion peaks and corresponding overlapping MRMtransitions ensure that product ion peaks near the end of the previousgroup are fully defined. Another method of ensuring that product ionpeaks near the end of the previous group are fully defined is to simplyallow two groups to be monitored at any one time, for example.

Groups are monitored or not monitored based on sentinel transitions. Ineach group at least one sentinel transition is selected from the MRMtransitions in the group. Any transition of the MRM transitions in agroup can be selected as the at least one sentinel transition. Forexample, the at least one sentinel transition can be selected from thebeginning, middle, or end of the group. In addition, more than onesentinel transition can be selected for each group, but each group thathas a following group must have at least one sentinel transition. Notethe last group in the series need not have a sentinel transition.

Also, different types of sentinel transitions can be used. The primarytype of sentinel transition is an MRM transition that identifies ortriggers the next group for monitoring. This type of sentinel transitionis, for example, a start sentinel transition. In various embodiments,another type of sentinel transition is a stop sentinel transition. Thistype of sentinel transition is not necessary, but can be used to triggerthe removal of a previous group from monitoring, for example. Stopsentinel transitions are discussed more below.

In plot 200, peak 221 is selected as the at least one sentineltransition for group 211, peak 222 is selected as the at least onesentinel transition for group 212, and peak 223 is selected as the atleast one sentinel transition for group 213, for example. Therefore, theMRM transition corresponding to peak 221 identifies or is used totrigger the monitoring of group 212. The MRM transition corresponding topeak 222 identifies or is used to trigger the monitoring of group 213.If there were a next group after group 213, the MRM transitioncorresponding to peak 223 would identify or would be used to trigger themonitoring of that group.

Once a group of MRM transitions is triggered it should not be monitoredfor the entire acquisition. Otherwise the number of MRM transitionsmonitored at any one time would not be limited near the end of theacquisition if no groups of transitions were removed. As describedabove, once at least one sentinel transition in the next contiguousgroup is detected, the transitions in the previous group can be removedfrom monitoring.

For example, the MRM transitions corresponding to the product ion peaksof group 211 are monitored first. When peak 221 corresponding to the atleast one sentinel transition of group 211 is detected above a certainthreshold intensity, the MRM transitions of group 212 are triggered formonitoring. As a result, for some time the MRM transitions correspondingto groups 211 and 212 are monitored together. However, when peak 222corresponding to the at least one sentinel transition of group 212 isdetected above a certain threshold intensity, not only are the MRMtransitions of group 213 triggered for monitoring, but the MRMtransitions of group 211 are removed from monitoring. In this way, theat least one sentinel transition of group 212 corresponding to peak 222acts as both a start and stop sentinel. It acts as a start sentinel bytriggering the monitoring of the MRM transitions of group 213, and itacts as a stop sentinel by removing the monitoring of the MRMtransitions of group 211.

This scheme works well when the sentinel transition corresponding topeak 222 is near the end of group 212. If the sentinel transition had aretention time near the beginning of group 212, the MRM transitions ofgroup 211 might be removed before the MRM transitions near the end ofgroup 211 had fully defined their product ion peaks. Of course onesolution, as described above, is to include large overlap regionsbetween groups. Another solution is to always pick sentinels near theend of each group.

A third solution, alluded to above, is to have specifically defined stopsentinels. For example, group 212 can include a stop sentinel transitioncorresponding to peak 232. When peak 232 is detected above a certainintensity threshold level, the MRM transitions of group 211 are removedfrom monitoring. Using start and stop sentinels can further limit thenumber of MRM transitions monitored over the entire acquisition. Forexample, during the time period between peak 232 and peak 222 only theMRM transitions of group 212 are monitored.

Using an MRM transition to trigger a group of other MRM transitions hasbeen described previously. For example, U.S. Pat. No. 8,026,479(hereinafter the “479 Patent”) describes using an MRM transition totrigger a group of confirmatory MRM transitions. In other words, then atriggering MRM transition is detected, a group of one or more otherconfirmatory MRM transitions expected at the same retention time aretriggered to confirm the presence of the compound represented by thetriggering MRM transition. The confirmatory MRM transitions may includedifferent precursor ions, but they are for the same target compoundtriggering MRM transition. In other words, the confirmatory MRMtransitions are selected for the same elution peaks as the triggeringMRM transition.

In contrast, various embodiments described herein use a triggering MRMtransition, called a sentinel transition, to trigger a group of MRMtransitions that include at least one transition targeting a retentiontime and an elution peak other than the retention time and elution peakof the sentinel transition. In other words, the triggering MRMtransition of the '479 Patent is designed to trigger a group oftransitions to re-interrogate the same elution peak or target compound,while the various embodiments described herein use a sentinel transitionto trigger a group of transitions that are aimed at interrogating atleast one later elution peak or target compound.

Therefore, a triggering MRM transition of the '479 Patent does nottrigger a next group of transitions from a contiguous series of groupsof transitions with different retention time ranges. Instead, atriggering MRM transition of the '479 Patent triggers a group of MRMtransitions with the same retention time as the triggering MRMtransition. The triggering MRM transition of the '479 Patent is also notpart of a group that precedes the triggered group in a series ofcontiguous groups.

Likewise, a sentinel transition of various embodiments described hereindoes not trigger a group of MRM transitions that all target the sameretention time as the sentinel transition. Instead, a sentineltransition of various embodiments described herein triggers a group ofMRM transitions that includes at least one MRM transition that targets aretention time or elution peak other than the retention time or elutionpeak targeted by the sentinel transition.

As described above, because the groups of transitions of variousembodiments described herein are not dependent on a specific retentiontime, workflows based on these systems and methods can be used withoutmodification whenever the separation device changes or the gradient ofseparation changes. The gradient of separation determines how fast orhow slow the targeted compounds of a sample are eluted. If the slope ofthe gradient of separation is increased, the targeted compounds of asample are eluted faster, for example.

FIG. 3 is an exemplary plot 300 of a gradient of separation, inaccordance with various embodiments. Plot 300 describes how thepercentage of organic composition of a chromatographic separation deviceis designed to vary over time. Plot 300 shows that the organiccomposition varies from 5 to 40% over 15 minutes producing slope 310.

FIG. 4 is an exemplary TIC chromatogram plot 400 showing the product ionpeaks produced by a method that used six sentinel transitions to triggerother groups of transitions for a sample that was separated according tothe gradient of separation shown in FIG. 3, in accordance with variousembodiments. The locations of the six sentinel transitions are indicatedby circles 410 in plot 400.

FIG. 5 is an exemplary plot 500 of a gradient of separation that has anincreased slope in comparison to the gradient of separation shown inFIG. 3, in accordance with various embodiments. Plot 500 describes howthe percentage of organic composition of a chromatographic separationdevice is designed to vary over time. Plot 500 shows that the organiccomposition varies from 5 to 40% over 8 minutes producing slope 510. Acomparison of slope 510 with slope 310 of FIG. 3 shows that slope 510 ofFIG. 5 is larger than slope 310 of FIG. 3. In other words, the elutiondescribed in FIG. 5 is much faster than the elution in FIG. 3.

FIG. 6 is an exemplary TIC chromatogram plot 600 showing the product ionpeaks produced by a method that used the same six sentinel transitionsusing to produce FIG. 4 to trigger other groups of transitions for asample that was separated according to the gradient of separation shownin FIG. 5, in accordance with various embodiments. The locations of thesix sentinel transitions are indicated by circles 610 in plot 600.

A comparison of FIGS. 4 and 6 shows that as the gradient of separationincreases, the product ion peaks are shifted to earlier times. Forexample, product ion peak 420 in FIG. 4 corresponds to product ion peak620 in FIG. 6. Product ion peak 420 in FIG. 4 occurs at a retention timeof about 12.5 min. Product ion peak 620 in FIG. 6 occurs at a retentiontime of about 8.0 min.

As described above, using scheduled MRM, the retention times of all ofthe MRMs would have to be changed when the gradient of separation isincreased from the gradient shown in FIG. 3 to the gradient shown inFIG. 5. Otherwise, for example, product ion peak 620 in FIG. 6 would notbe found, because the scheduled MRM would be looking for product ionpeak 620 at a retention time of 12.5 min.

FIGS. 4 and 6 show that the grouping of MRM transitions by the order ofretention time and the use of sentinel transitions allows all production peaks to be found even if the gradient of separation increases. Inaddition, no re-computation of retention time is need for eachtransition.

System for Triggering a Group of Contiguous Groups of MRM Transitions

FIG. 7 is a schematic diagram of system 700 for triggering a group ofmultiple reaction monitoring (MRM) transitions from a series ofcontiguous groups when at least one sentinel transition of the group isdetected as part of a previous group, in accordance with variousembodiments. System 700 includes separation device 740, ion source 710,tandem mass spectrometer 720, and processor 730. Separation device 740separates one or more compounds from a sample. The sample is a samplemixture, for example. Separation device 740 can separate compound overtime using one of a variety of techniques. These techniques include, butare not limited to, ion mobility, gas chromatography (GC), liquidchromatography (LC), capillary electrophoresis (CE), or flow injectionanalysis (FIA).

Ion source 710 can be part of tandem mass spectrometer 720, or can be aseparate device. Ion source 710 ionizes the separated one or morecompounds received from separation device 740, producing an ion beam ofone or more precursor ions.

Tandem mass spectrometer 720 can include, for example, one or morephysical mass filters and one or more physical mass analyzers. A massanalyzer of tandem mass spectrometer 720 can include, but is not limitedto, a time-of-flight (TOF), quadrupole, an ion trap, a linear ion trap,an orbitrap, or a Fourier transform mass analyzer.

Tandem mass spectrometer 720 receives the ion beam from ion source 710.For each cycle of a plurality cycles, tandem mass spectrometer 720executes on the ion beam a series of MRM precursor ion to product iontransitions read from a list. The list is a duty cycle list, forexample. For each transition of the series, tandem mass spectrometer 720selects and fragments a precursor ion of each transition and massanalyzes a small mass-to-charge ratio (m/z) range around the m/z of aproduct ion of the each transition to determine if the product ion ofthe transition is detected.

Processor 730 can be, but is not limited to, a computer, microprocessor,or any device capable of sending and receiving control signals and datafrom tandem mass spectrometer 720 and processing data. Processor 730 canbe, for example, computer system 100 of FIG. 1. In various embodiments,processor 730 is in communication with tandem mass spectrometer 720 andseparation device 740.

Processor 730 receives a plurality of MRM transitions to be used tointerrogate the sample. The plurality of MRM transitions are receivedfrom a user, for example. Processor 730 divides the plurality of MRMtransitions into two or more contiguous groups of MRM transitions sothat different groups can be monitored separately during the pluralityof cycles. For example, processor 730 can order the plurality of MRMtransitions according to expected retention time. Expected retentiontimes are received for each MRM transition from a user, for example.Processor 730 can then divide the ordered MRM transitions into two ormore contiguous groups of MRM transitions so that different groups canbe monitored separately during the plurality of cycles.

Processor 730 selects at least one sentinel transition in each group ofthe two or more contiguous groups that identifies a next group of thetwo or more contiguous groups that is to be monitored. The at least onesentinel transition identifies or triggers the next adjacent group ofthe two or more contiguous groups, for example.

Processor 730 places a first group of the two or more contiguous groupson the list of tandem mass spectrometer 720, so that tandem massspectrometer 720 monitors the MRM transitions of the first group. Whenat least one sentinel transition of the first group is detected bytandem mass spectrometer 720, processor 730 places a next group of thetwo or more contiguous groups identified by the sentinel transition onthe list.

In various embodiments, each group of the two or more contiguous groupsincludes MRM transitions that overlap with MRM transitions of at leastone other group of the two or more groups in order to ensure correctpeak definition. The overlap is with an adjacent group, for example.

In various embodiments, processor 730 further removes the first groupfrom the list, when a sentinel transition of the next group is detected.

In various embodiments, processor 730 further selects a stop sentineltransition for each group of the two or more contiguous groups thatidentifies a previous group of the two or more contiguous groups. When astop sentinel transition of a group is detected, processor 730 furtherremoves a previous group identified by the stop sentinel from the list.

In various embodiments, the sentinel transitions for each group of thetwo or more contiguous groups are monitored as part of each group, orfor the entire acquisition. For example, each group of the two or morecontiguous groups further includes each sentinel transition of the othergroups of the two or more contiguous groups. This allows sentineltransitions to be independent of retention windows also. As a result,tandem mass spectrometer 720 detects a product ion of the eachtransition without using a retention time window for the eachtransition.

Alternatively, sentinel transitions can be monitored with wide retentiontime windows. The groups MRM transitions triggered by sentineltransitions, however, are not monitored according to retention timewindows.

Processor 730 can select any of the MRM transitions of a group as the atleast one sentinel. For example, processor 730 can select the at leastone sentinel transition in each group by selecting an MRM transition ofeach group with the latest expected retention time. In other words,processor 730 can select the MRM transition at the end of each group asthe sentinel transition.

In various embodiments, an MRM transitions can include a precursor ionand/or one or more product ions of the precursor ion that are found in afull scan product ion spectrum. For example, for TOF and orbitrap massanalyzers, tandem mass spectrometer 720 executes on the ion beam aseries of precursor ion to full product ion spectrum scans read from alist. The list is a duty cycle list, for example. For each precursor ionof the series, tandem mass spectrometer 720 selects and fragments aprecursor ion and mass analyzes an entire mass-to-charge ratio (m/z)range of product ions to determine if the precursor ion is detected. Theunfragmented precursor ion and/or one or more product ions of theprecursor ion are detected in the full product ion spectrum, forexample.

Processor 730 receives a plurality of precursor ions and/or one or moreproduct ions of the precursor ion to be used to interrogate the sample.The plurality of precursor ions and/or one or more product ions of theprecursor ion are received from a user, for example. Processor 730divides the plurality of precursor ions and/or one or more product ionsof the precursor ion into two or more contiguous groups of precursorions and/or one or more product ions of the precursor ion so thatdifferent groups can be monitored separately during the plurality ofcycles. For example, processor 730 can order the plurality of precursorions and/or one or more product ions of the precursor ion according toexpected retention time. Expected retention times are received for eachprecursor ion from a user, for example. Processor 730 can then dividethe ordered precursor ions and/or one or more product ions of theprecursor ion into two or more contiguous groups of precursor ionsand/or one or more product ions of the precursor ion so that differentgroups can be monitored separately during the plurality of cycles.

Processor 730 selects at least one sentinel precursor ion and/or one ormore sentinel product ions of the precursor ion in each group of the twoor more contiguous groups that identifies a next group of the two ormore contiguous groups that is to be monitored. The at least onesentinel precursor ion and/or one or more sentinel product ions of theprecursor ion identifies or triggers the next adjacent group of the twoor more contiguous groups, for example.

Processor 730 places a first group of the two or more contiguous groupson the list of tandem mass spectrometer 720, so that tandem massspectrometer 720 monitors the precursor ions and/or one or more productions of the precursor ion of the first group. When at least one sentinelprecursor ion of the first group is detected by tandem mass spectrometer720, processor 730 places a next group of the two or more contiguousgroups identified by the sentinel precursor ion and/or one or moresentinel product ions of the precursor ion on the list.

Method for Triggering a Group of Contiguous Groups of MRM Transitions

FIG. 8 is a flowchart showing a method 800 for triggering a group of MRMtransitions from a series of contiguous groups when at least onesentinel transition of the group is detected as part of a previousgroup, in accordance with various embodiments.

In step 810 of method 800, one or more compounds are separated from asample using a separation device.

In step 820, the separated one or more compounds received from theseparation device are ionized using an ion source, producing an ion beamof one or more precursor ions.

In step 830, the ion beam is received from the ion source using a tandemmass spectrometer and, for each cycle of a plurality cycles, a series ofMRM precursor ion to product ion transitions read from a list areexecuted on the ion beam using the tandem mass spectrometer. For eachtransition of the series, the tandem mass spectrometer selects andfragments a precursor ion of the each transition and mass analyzes asmall mass-to-charge ratio (m/z) range around the m/z of a product ionof the each transition to determine if the product ion of the eachtransition is detected.

In step 840, a plurality of MRM transitions to be used to monitor thesample are received using a processor.

In step 850, the plurality of MRM transitions are divided into two ormore contiguous groups of MRM transitions so that different groups canbe monitored separately during the plurality of cycles using theprocessor.

In step 860, at least one sentinel transition is selected in each groupof the two or more contiguous groups that identifies a next group of thetwo or more contiguous groups that is to be monitored using theprocessor.

In step 870, a first group of the two or more contiguous groups isplaced on the list of the tandem mass spectrometer using the processor.

In step 880, when at least one sentinel transition of the first group isdetected by the tandem mass spectrometer, a next group of the two ormore contiguous groups identified by the sentinel transition is placedon the list using the processor.

Computer Program Product for Triggering a Group of Contiguous Groups

In various embodiments, computer program products include a tangiblecomputer-readable storage medium whose contents include a program withinstructions being executed on a processor so as to perform a method fortriggering a group of MRM transitions from a series of contiguous groupswhen at least one sentinel transition of the group is detected as partof a previous group. This method is performed by a system that includesone or more distinct software modules.

FIG. 9 is a schematic diagram of a system 900 that includes one or moredistinct software modules that performs a method for triggering a groupof MRM transitions from a series of contiguous groups when at least onesentinel transition of the group is detected as part of a previousgroup, in accordance with various embodiments. System 900 includesmeasurement module 910 and analysis module 920.

For each cycle of a plurality cycles, measurement module 910 instructs atandem mass spectrometer to execute on an ion beam a series of MRMprecursor ion to product ion transitions read from a list. For eachtransition of the series, the tandem mass spectrometer selects andfragments a precursor ion of the each transition and mass analyzes asmall mass-to-charge ratio (m/z) range around the m/z of a product ionof the each transition to determine if the product ion of the eachtransition is detected. The ion beam is produced by an ion source thationizes one or more compounds separated from a sample using a separationdevice.

Analysis module 920 receives a plurality of MRM transitions to be usedto monitor the sample. Analysis module 920 divides the plurality of MRMtransitions into two or more contiguous groups of MRM transitions sothat different groups can be monitored separately during the pluralityof cycles. Analysis module 920 selects at least one sentinel transitionin each group of the two or more contiguous groups that identifies anext group of the two or more contiguous groups that is to be monitored.

Measurement module 910 places a first group of the two or morecontiguous groups on the list of the tandem mass spectrometer. When atleast one sentinel transition of the first group is detected by thetandem mass spectrometer, measurement module 910 places a next group ofthe two or more contiguous groups identified by the sentinel transitionon the list.

While the present teachings are described in conjunction with variousembodiments, it is not intended that the present teachings be limited tosuch embodiments. On the contrary, the present teachings encompassvarious alternatives, modifications, and equivalents, as will beappreciated by those of skill in the art.

Further, in describing various embodiments, the specification may havepresented a method and/or process as a particular sequence of steps.However, to the extent that the method or process does not rely on theparticular order of steps set forth herein, the method or process shouldnot be limited to the particular sequence of steps described. As one ofordinary skill in the art would appreciate, other sequences of steps maybe possible. Therefore, the particular order of the steps set forth inthe specification should not be construed as limitations on the claims.Similarly, though the described application used MRM as a detectiontechnique, the described method can be applied to any targeted analysisfor MS/MS analysis such as MRM3, single ion monitoring (SIM) or eventargeted product ion scan (TOF-MS). In addition, the claims directed tothe method and/or process should not be limited to the performance oftheir steps in the order written, and one skilled in the art can readilyappreciate that the sequences may be varied and still remain within thespirit and scope of the various embodiments.

What is claimed is:
 1. A system for triggering a group of multiplereaction monitoring (MRM) transitions from a series of contiguous groupswhen at least one sentinel transition of the group is detected as partof a previous group, comprising: a separation device that separates oneor more compounds from a sample; an ion source that ionizes theseparated one or more compounds received from the separation device,producing an ion beam of one or more precursor ions; a tandem massspectrometer that receives the ion beam from the ion source and for eachcycle of a plurality cycles executes on the ion beam a series of MRMprecursor ion to product ion transitions read from a list, wherein foreach transition of the series, the tandem mass spectrometer selects andfragments a precursor ion of the each transition and mass analyzes asmall mass-to-charge ratio (m/z) range around the m/z of a product ionof the each transition to determine if the product ion of the eachtransition is detected; and a processor in communication with the tandemmass spectrometer that receives a plurality of MRM transitions to beused to monitor the sample, divides the plurality of MRM transitionsinto two or more contiguous groups of MRM transitions so that differentgroups can be monitored separately during the plurality of cycles,selects at least one sentinel transition in each group of the two ormore contiguous groups that identifies a next group of the two or morecontiguous groups that is to be monitored, places a first group of thetwo or more contiguous groups on the list of the tandem massspectrometer, and when at least one sentinel transition of the firstgroup is detected by the tandem mass spectrometer, places a next groupof the two or more contiguous groups identified by the sentineltransition on the list.
 2. The system of claim 1, wherein each group ofthe two or more contiguous groups includes MRM transitions that overlapwith MRM transitions of at least one other group of the two or moregroups in order to ensure correct peak definition.
 3. The system ofclaim 1, wherein the processor further removes the first group from thelist, when a sentinel transition of the next group is detected by thetandem mass spectrometer.
 4. The system of claim 1, wherein theprocessor further selects a stop sentinel transition for each group ofthe two or more contiguous groups that identifies a previous group ofthe two or more contiguous groups, and when a stop sentinel transitionof an group is detected, the processor further removes a previous groupidentified by the stop sentinel from the list.
 5. The system of claim 1,wherein each group of the two or more contiguous groups further includeseach sentinel transition of the other groups of the two or morecontiguous groups.
 6. The system of claim 1, wherein the tandem massspectrometer detects a product ion of the each transition without usinga retention time window for the each transition.
 7. The system of claim1, wherein the processor selects the at least one sentinel transition ineach group by selecting an MRM transition of the each group with thelatest expected retention time.
 8. A method for triggering a group ofmultiple reaction monitoring (MRM) transitions from a series ofcontiguous groups when at least one sentinel transition of the group isdetected as part of a previous group, comprising: separating one or morecompounds from a sample using a separation device; ionizing theseparated one or more compounds received from the separation deviceusing an ion source, producing an ion beam of one or more precursorions; receiving the ion beam from the ion source using a tandem massspectrometer and, for each cycle of a plurality cycles, executing on theion beam a series of MRM precursor ion to product ion transitions readfrom a list using the tandem mass spectrometer, wherein for eachtransition of the series, the tandem mass spectrometer selects andfragments a precursor ion of the each transition and mass analyzes asmall mass-to-charge ratio (m/z) range around the m/z of a product ionof the each transition to determine if the product ion of the eachtransition is detected; receiving a plurality of MRM transitions to beused to monitor the sample using a processor; dividing the plurality ofMRM transitions into two or more contiguous groups of MRM transitions sothat different groups can be monitored separately during the pluralityof cycles using the processor; selecting at least one sentineltransition in each group of the two or more contiguous groups thatidentifies a next group of the two or more contiguous groups that is tobe monitored using the processor; placing a first group of the two ormore contiguous groups on the list of the tandem mass spectrometer usingthe processor; and when at least one sentinel transition of the firstgroup is detected by the tandem mass spectrometer, placing a next groupof the two or more contiguous groups identified by the sentineltransition on the list using the processor.
 9. The method of claim 8,wherein each group of the two or more contiguous groups includes MRMtransitions that overlap with MRM transitions of at least one othergroup of the two or more groups in order to ensure correct peakdefinition.
 10. The method of claim 8, further comprising when asentinel transition of the next group is detected by the tandem massspectrometer, removing the first group from the list using theprocessor.
 11. The method of claim 8, further comprising selecting astop sentinel transition for each group of the two or more contiguousgroups that identifies a previous group of the two or more contiguousgroups using the processor, and when a stop sentinel transition of angroup is detected, further removing a previous group identified by thestop sentinel from the list using the processor.
 12. The method of claim8, wherein each group of the two or more contiguous groups furtherincludes each sentinel transition of the other groups of the two or morecontiguous groups.
 13. The method of claim 8, wherein a product ion ofthe each transition is detected without using a retention time windowfor the each transition using the tandem mass spectrometer.
 14. Themethod of claim 8, further comprising selecting the at least onesentinel transition in each group by selecting an MRM transition of theeach group with the latest expected retention time.
 15. A computerprogram product, comprising a non-transitory and tangiblecomputer-readable storage medium whose contents include a program withinstructions being executed on a processor so as to perform a method fortriggering a group of multiple reaction monitoring (MRM) transitionsfrom a series of contiguous groups when at least one sentinel transitionof the group is detected as part of a previous group, comprising:providing a system, wherein the system comprises one or more distinctsoftware modules, and wherein the distinct software modules comprise ameasurement module and an analysis module; for each cycle of a pluralitycycles, instructing a tandem mass spectrometer to execute on an ion beama series of MRM precursor ion to product ion transitions read from alist using the measurement module, wherein for each transition of theseries, the tandem mass spectrometer selects and fragments a precursorion of the each transition and mass analyzes a small mass-to-chargeratio (m/z) range around the m/z of a product ion of the each transitionto determine if the product ion of the each transition is detected andwherein the ion beam is produced by an ion source that ionizes one ormore compounds separated from a sample using a separation device;receiving a plurality of MRM transitions to be used to monitor thesample using the analysis module; dividing the plurality of MRMtransitions into two or more contiguous groups of MRM transitions sothat different groups can be monitored separately during the pluralityof cycles using the analysis module; selecting at least one sentineltransition in each group of the two or more contiguous groups thatidentifies a next group of the two or more contiguous groups that is tobe monitored using the analysis module; placing a first group of the twoor more contiguous groups on the list of the tandem mass spectrometerusing the measurement module; and when at least one sentinel transitionof the first group is detected by the tandem mass spectrometer, placinga next group of the two or more contiguous groups identified by thesentinel transition on the list using the measurement module.